Epoxidation using peroxygenase

ABSTRACT

The invention relates to enzymatic methods for epoxidation of a non-cyclic aliphatic alkene, or a terpene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/021,544filed on Jun. 28, 2018, now pending, which is a divisional of U.S.application Ser. No. 15/876,974 filed on Jan. 22, 2018, now U.S. Pat.No. 10,017,483, which is a divisional of U.S. application Ser. No.15/497,548 filed on Apr. 26, 2017, now U.S. Pat. No. 9,908,860, which isa divisional of U.S. application Ser. No. 15/250,290 filed on Aug. 29,2016, now U.S. Pat. No. 9,663,806, which is a divisional of U.S.application Ser. No. 14/382,957, now U.S. Pat. No. 9,458,478, which is a35 U.S.C. 371 national application of international application no.PCT/EP2013/056326 filed on Mar. 25, 2013, which claims priority or thebenefit under 35 U.S.C. 119 of European application nos. 12162791.3 and12165214.3 filed on Mar. 31, 2012 and Apr. 23, 2012, respectively, andU.S. provisional application Nos. 61/622,686 and 61/636,956 filed onApr. 11, 2012 and Apr. 23, 2012, respectively. The content of theseapplications is fully incorporated herein by reference.

CROSS-REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to use of peroxygenases for epoxidation ofnon-cyclic aliphatic alkenes, or terpenes.

Background

A peroxygenase denoted AaP from the agaric basidiomycete strain Agrocybeaegerita (strain TM-A1) was found to oxidize aryl alcohols andaldehydes. The AaP peroxygenase was purified from A. aegerita TM A1 byseveral steps of ion chromatography, the molecular weight was determinedby SDS-PAGE and the N-terminal 14 amino acid sequence was determinedafter 2-D electrophoresis but the encoding gene was not isolated(Ullrich et al., 2004, Appl. Env. Microbiol. 70(8): 4575-4581).

WO 2006/034702 discloses methods for the enzymatic hydroxylation ofnon-activated hydrocarbons, such as, naphtalene, toluol and cyclohexane,using the AaP peroxygenase enzyme of Agrocybe aegerita TM A1. This isalso described in Ullrich and Hofrichter, 2005, FEBS Letters 579:6247-6250.

WO 2008/119780 discloses eight different peroxygenases from Agrocybeaegerita, Coprinopsis cinerea, Laccaria bicolor and Coprinus radians.

DE 103 32 065 A1 discloses methods for the enzymatic preparation ofacids from alcohols through the intermediary formation of aldehydes byusing the AaP peroxygenase enzyme of Agrocybe aegerita TM A1.

A method was reported for the rapid and selective spectrophotometricdirect detection of aromatic hydroxylation by the AaP peroxygenase(Kluge et al., 2007, Appl. Microbiol. Biotechnol. 75: 1473-1478).

It is well-known that a direct regioselective introduction of oxygenfunctions (oxygenation) into organic molecules constitutes a problem inchemical synthesis. The products may be used as important intermediatesin a wide variety of different syntheses.

It is known that an intracellular enzyme, methane monooxygenase (MMO, EC14.13.25), oxygenates/hydroxylates the terminal carbon of somehydrocarbons. The MMO enzyme consists of several protein components andis solely formed by methylotrophic bacteria (e.g. Methylococcuscapsulatus); it requires complex electron donors such as NADH or NADPH,auxiliary proteins (flavin reductases, regulator protein) and molecularoxygen (O₂). The natural substrate of MMO is methane, which is oxidizedto methanol. As a particularly unspecific biocatalyst, MMOoxygenates/hydroxylates, as well as methane, a series of furthersubstrates such as n-alkanes and their derivatives, cycloalkanes,aromatics, carbon monoxide and heterocycles. Utilization of the enzymein biotechnology is currently not possible, since it is difficult toisolate, like most intracellular enzymes, it is of low stability, andthe cosubstrates required are relatively expensive.

SUMMARY OF THE INVENTION

In a first aspect, the inventors of the present invention have providedan enzymatic method for producing an epoxide, comprising contacting anon-cyclic aliphatic alkene, or a terpene, with hydrogen peroxide and aperoxygenase; wherein the peroxygenase comprises an amino acid sequencewhich has at least 60% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In an embodiment, theamino acid sequence comprises the motif: E-H-D-[G,A]-S-[L,I]-S-R (SEQ IDNO: 21).

Definitions

Peroxygenase activity: The term “peroxygenase activity” means an“unspecific peroxygenase” activity according to EC 1.11.2.1, thatcatalyzes insertion of an oxygen atom from H₂O₂ into a variety ofsubstrates, such as nitrobenzodioxole. For purposes of the presentinvention, peroxygenase activity is determined according to theprocedure described in Poraj-Kobielska et al., 2012, “Aspectrophotometric assay for the detection of fungal peroxygenases”,Analytical Biochemistry 421(1): 327-329.

The peroxygenase of the present invention has at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the peroxygenase activity of the maturepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having peroxygenase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In a preferred aspect, the mature polypeptide hasthe amino acid sequence shown in positions 1 to 328 of SEQ ID NO:1 basedon the N-terminal peptide sequencing data (Ullrich et al., 2004, Appl.Env. Microbiol. 70(8): 4575-4581), elucidating the start of the matureprotein of AaP peroxygenase enzyme.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277; emboss.org), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra; emboss.org), preferably version 3.0.0 or later. The optionalparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitutionmatrix. The output of Needle labeled “longest identity” (obtained usingthe -nobrief option) is used as the percent identity and is calculatedas follows: (Identical Deoxyribonucleotides×100)/(Length ofAlignment−Total Number of Gaps in Alignment).

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20; or a homologous sequence thereof; as well as geneticmanipulation of the DNA encoding such a polypeptide. The modificationcan be a substitution, a deletion and/or an insertion of one or more(several) amino acids as well as replacements of one or more (several)amino acid side chains.

DETAILED DESCRIPTION OF THE INVENTION Peroxygenase

The present invention relates to methods for epoxidation using apolypeptide, which is preferably recombinantly produced, havingperoxygenase activity (referenced as “peroxygenase”), which comprises orconsists of an amino acid sequence having at least 60% identity,preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably SEQ ID NO: 1.

In a preferred embodiment, the peroxygenase comprises an amino acidsequence represented by the motif: E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO:21).

In yet another embodiment, the polypeptide of the first aspect comprisesor consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably SEQID NO: 1; or a fragment thereof having peroxygenase activity; preferablythe polypeptide comprises or consists of the mature polypeptide of SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20; preferably SEQ ID NO: 1.

Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,peroxygenase activity) to identify amino acid residues that are criticalto the activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably SEQ IDNO: 1; is 10, preferably 9, more preferably 8, more preferably 7, morepreferably at most 6, more preferably 5, more preferably 4, even morepreferably 3, most preferably 2, and even most preferably 1.

Hydrogen Peroxide

The hydrogen peroxide required by the peroxygenase may be provided as anaqueous solution of hydrogen peroxide or a hydrogen peroxide precursorfor in situ production of hydrogen peroxide. Any solid entity whichliberates upon dissolution a peroxide, which is useable by peroxygenase,can serve as a source of hydrogen peroxide. Compounds which yieldhydrogen peroxide upon dissolution in water or an appropriate aqueousbased medium include but are not limited to metal peroxides,percarbonates, persulphates, perphosphates, peroxyacids, alkyperoxides,acylperoxides, peroxyesters, urea peroxide, perborates andperoxycarboxylic acids or salts thereof.

Another source of hydrogen peroxide is a hydrogen peroxide generatingenzyme system, such as an oxidase together with a substrate for theoxidase. Examples of combinations of oxidase and substrate comprise, butare not limited to, amino acid oxidase (see e.g. U.S. Pat. No.6,248,575) and a suitable amino acid, glucose oxidase (see e.g. WO95/29996) and glucose, lactate oxidase and lactate, galactose oxidase(see e.g. WO 00/50606) and galactose, and aldose oxidase (see e.g. WO99/31990) and a suitable aldose.

By studying EC 1.1.3._, EC 1.2.3._, EC 1.4.3._, and EC 1.5.3._(—) orsimilar classes (under the International Union of Biochemistry), otherexamples of such combinations of oxidases and substrates are easilyrecognized by one skilled in the art.

Alternative oxidants which may be applied for peroxygenases may beoxygen combined with a suitable hydrogen donor like ascorbic acid,dehydroascorbic acid, dihydroxyfumaric acid or cysteine. An example ofsuch oxygen hydrogen donor system is described by Pasta et al., 1999,Biotechnology & Bioengineering 62(4): 489-493.

Hydrogen peroxide or a source of hydrogen peroxide may be added at thebeginning of or during the method of the invention, e.g. as one or moreseparate additions of hydrogen peroxide; or continuously as fed-batchaddition. Typical amounts of hydrogen peroxide correspond to levels offrom 0.001 mM to 25 mM, preferably to levels of from 0.005 mM to 5 mM,and particularly to levels of from 0.01 to 1 mM or 0.02 to 2 mM hydrogenperoxide. Hydrogen peroxide may also be used in an amount correspondingto levels of from 0.1 mM to 25 mM, preferably to levels of from 0.5 mMto 15 mM, more preferably to levels of from 1 mM to 10 mM, and mostpreferably to levels of from 2 mM to 8 mM hydrogen peroxide.

Surfactants

The method of the invention may include application of a surfactant (forexample, as part of a detergent formulation or as a wetting agent).Surfactants suitable for being applied may be non-ionic (includingsemi-polar), anionic, cationic and/or zwitterionic; preferably thesurfactant is anionic (such as linear alkylbenzenesulfonate,alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcoholethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methylester, alkyl- or alkenylsuccinic acid or soap) or non-ionic (such asalcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acylN-alkyl derivatives of glucosamine (“glucamides”)), or a mixturethereof.

When included in the method of the invention, the concentration of thesurfactant will usually be from about 0.01% to about 10%, preferablyabout 0.05% to about 5%, and more preferably about 0.1% to about 1% byweight.

Aliphatic Alkene

The aliphatic alkene (unsaturated aliphatic hydrocarbon), which isepoxidized in the method of the invention, is a non-cyclic aliphaticalkene, which is linear or branched; and substituted or unsubstituted.Preferably, the aliphatic alkene is unsubstituted. Branched alkenescorrespond to isomers of linear alkenes.

In an embodiment, the non-cyclic aliphatic alkene has at least threecarbons. In another embodiment, the aliphatic alkene has a carbon-carbondouble bond (an unsaturated carbon) at one end.

Preferably, the aliphatic alkene is propene, butene, pentene, hexene,heptene, octene, nonene, decene, undecene, dodecene, tridecene,tetradecene, pentadecene or hexadecene, or isomers thereof. Morepreferably, the aliphatic akene is propene, 1-butene, 1-pentene,1-hexene, 2-hexene, 3-hexene, 1-heptene, 1-octene, 2-methyl-2-butene,2,3-dimethyl-2-butene, cis/trans-2-butene, isobutene, 1,3-butadiene, andisoprene; or isomers thereof.

When the aliphatic alkenes are substituted (functional groups attached),the preferred substituents are halogen, hydroxyl, carboxyl, amino,nitro, cyano, thiol, sulphonyl, formyl, acetyl, methoxy, ethoxy,carbamoyl and sulfamoyl; more preferred substituents are chloro,hydroxyl, carboxyl and sulphonyl; and most preferred substituents arechloro and carboxyl.

The aliphatic alkenes may be substituted by up to 10 substituents, up to8 substituents, up to 6 substituents, up to 4 substituents, up to 2substituents, or by up to one substituent.

Terpene

The terpenes, which are epoxidized according to the invention, includeisoprene, and compounds having multiples of the isoprene structure. Theterpenes of the invention also include terpenoids.

Terpenes can be subdivided in monoterpenes (two isoprene units),sesquiterpenes (three isoprene units), diterpenes (four isoprene units),triterpenes (six isoprene units), tetraterpenes (eight isoprene units)etc. Terpenes can be monocyclic, bicyclic, tricyclic, etc.

Examples of monoterpenes include geraniol (rose oil) and monocyclicmonoterpenes, such as (−)-menthol (peppermint oil), R-(+)-carvone(caroway oil), S-(−)-carvone (spearmint oil), R-(+)-limonene (orangeoil), and S-(−)-limonene (pine oil smell).

Preferably the terpene of the invention is isoprene or a monoterpene;more preferably the terpene is a cyclic terpene, such as a monocyclicmonoterpene, such as limonene.

Terpenes and cyclic terpenes like limonene are found broadly in nature.Limonene is the most abundant common naturally occurringterpene—produced by more than 300 plants and is a major component incitrus peel oil. Epoxidation of limonene is of particular relevancesince it forms the basis for the synthesis of fragrances and drugs.Peroxygenases epoxidize limonene both in the ring position and in theside chain—with dominating preference for the ring position (see Example1).

Methods and Uses

The present invention provides a method for producing an epoxide from anon-cyclic aliphatic alkene, or a terpene (a method for epoxidation of anon-cyclic aliphatic alkene or a terpene), comprising contacting thealiphatic alkene, or terpene, with a peroxygenase and hydrogen peroxide.Thus, the invention provides a method for converting a non-cyclicaliphatic alkene, or terpene, to an epoxide, by oxidation of thealiphatic alkene or terpene at a carbon-carbon double bond.

The aliphatic alkene includes at least three carbons. Preferably, thealiphatic alkene has a carbon-carbon double bond (an unsaturated carbon)at one end.

Accordingly, in a first aspect, the present invention provides a methodfor producing an epoxide, comprising contacting a non-cyclic aliphaticalkene, or terpene, with hydrogen peroxide and a peroxygenase; whereinthe peroxygenase comprises an amino acid sequence which has at least 60%identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20. The aliphatic alkene may be substituted orunsubstituted, linear or branched.

In an embodiment, the amino acid sequence comprises the motif:E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO: 21).

In an embodiment, the peroxygenase comprises or consists of an aminoacid sequence having at least 65% identity, preferably at least 70%identity, more preferably at least 75% identity, more preferably atleast 80% identity, more preferably at least 85% identity, mostpreferably at least 90% identity, and in particular at least 95%identity to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably SEQ IDNO: 1. In a preferred embodiment, the peroxygenase comprises or consistsof the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably SEQ ID NO: 1; or afragment thereof having peroxygenase activity.

In an embodiment, the terpene is isoprene or a monoterpene; preferablythe terpene is a cyclic terpene, such as a monocyclic monoterpene, suchas limonene.

In another embodiment, the aliphatic alkene has one or more substituentsselected from the group consisting of halogen, hydroxyl, carboxyl,amino, nitro, cyano, thiol, sulphonyl, formyl, acetyl, methoxy, ethoxy,carbamoyl and sulfamoyl. Preferably, the substituent(s) are selectedfrom the group consisting of chloro, hydroxyl, carboxyl and sulphonyl;in particular, chloro and carboxyl.

In another embodiment, the aliphatic alkene consists of at least threecarbons, and has a carbon-carbon double bond at one end.

In another embodiment, the aliphatic alkene is propene, butene, pentene,hexene, heptene, octene, nonene, decene, undecene, dodecene, tridecene,tetradecene, pentadecene or hexadecene, or isomers thereof. In a morepreferred embodiment, the aliphatic alkene is propene, 1-butene,1-pentene, 1-hexene, 2-hexene, 3-hexene, 1-heptene, 1-octene,2-methyl-2-butene, 2,3-dimethyl-2-butene, cis/trans-2-butene, isobutene,1,3-butadiene, and isoprene; or isomers thereof.

In another embodiment, the aliphatic alkene is unsubstituted.

In another embodiment, the aliphatic alkene is linear.

Preferred epoxides, which can be produced by the method of the inventioninclude, but are not limited to, propylene oxide (1,2-epoxypropane) andepoxy terpenes (including epoxy terpenoids), such as limonene oxide; and2-methyloxirane, 2-ethyloxirane, 2-propyloxirane, 2-butyloxirane,2-pentyloxirane, 2-hexyloxirane, 2,2,3-trimethyloxirane,2,2,3,3-tetramethyloxirane, (2S,3S)-2,3-dimethyloxirane,(2R,3S)-2,3-Dimethyloxirane, 2,2-dimethyloxirane,2-methyl-2-(4-methylcyclohex-3-en-1-yl)oxirane,2-methyl-2-(4-methylcyclohex-3-en-1-yl)oxirane, 2-ethenyloxirane,2-ethenyl-2-methyloxirane, 2-methyl-3-propyloxirane, and2,3-diethyloxirane.

The method of the invention may be used for a variety of purposes, likebulk chemical synthesis (biocatalysis). Alkenes are efficientlyepoxidized by peroxygenases. Propylene oxide is a highly reactivesubstance and one of the most important chemical intermediates. It isthe starting material for a broad spectrum of products, includingpolymers (polyurethanes, polyesters), oxygenated solvents (propyleneglycol ethers) and industrial fluids (monopropylene glycol andpolyglycols). Annually 6,500,000 MT are produced. Other longer chainalkenes including branched alkenes are epoxidized in a similarmanner—including butene and isobutene.

Polymerization of an epoxide gives a polyether, for example ethyleneoxide polymerizes to give polyethylene glycol, also known aspolyethylene oxide.

The methods of the invention may be carried out with an immobilizedperoxygenase.

The methods of the invention may be carried out in an aqueous solvent(reaction medium), various alcohols, ethers, other polar or non-polarsolvents, or mixtures thereof. By studying the characteristics of thealiphatic hydrocarbon used in the methods of the invention, suitableexamples of solvents are easily recognized by one skilled in the art. Byraising or lowering the pressure at which the oxidation is carried out,the solvent (reaction medium) and the aliphatic hydrocarbon can bemaintained in a liquid phase at the reaction temperature.

The methods according to the invention may be carried out at atemperature between 0 and 90° C., preferably between 5 and 80° C., morepreferably between 10 and 70° C., even more preferably between 15 and60° C., most preferably between 20 and 50° C., and in particular between20 and 40° C.

The methods of the invention may employ a treatment time of from 10seconds to (at least) 24 hours, preferably from 1 minute to (at least)12 hours, more preferably from 5 minutes to (at least) 6 hours, mostpreferably from 5 minutes to (at least) 3 hours, and in particular, from5 minutes to (at least) 1 hour.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

The amino acid sequence of the peroxygenase from Agrocybe aegerita isshown as SEQ ID NO: 1.

Example 1 Alkene Epoxidation Catalyzed by a Fungal Peroxygenase Reagents

Commercially available chemicals were purchased from Sigma-Aldrich, TCIEurope and Chemos GmbH, except for 2,3-epoxy-2-methylbutane which waspurchased from Acros Organics. The extracellular peroxygenase ofAgrocybe aegerita (wild type, isoform II, 44 kDa) was produced andpurified as described previously. The enzyme preparation was homogeneousby SDS polyacrylamide gel electrophoresis and exhibited an A₄₁₈/A₂₈₀ratio of 1.86. The specific activity of the peroxygenase was 63 U mg⁻¹,where 1 U represents the oxidation of 1 μmol of 3,4-dimethoxybenzylalcohol to 3,4-dimethoxybenzaldehyde in 1 min at 23° C.

Reaction Conditions

Typical reaction mixtures (total volume: 0.2 ml; 1.2 ml for gaseousalkenes) contained purified peroxygenase (1-2 U ml⁻¹, 0.38-0.76 μM)dissolved in potassium phosphate buffer (10 mM, pH 7.0), acetone (60%,pH 5.3), and the alkene substrate (5% vol/vol; except for +/−limonene 5mM of the substrate was used). The reactions were started by theaddition of H₂O₂ via a syringe pump (4 mM h⁻¹, except for2-methyl-2-butene and 2,3-dimethyl-2-butene 2 mM h⁻¹ was used), stirredat room temperature for 30 min, and stopped at which timechromatographic analyses showed that product formation was complete.Gaseous propene and n-butene were treated under the same conditions butby continuously bubbling the pure gas through the reaction vial (approx.11 h⁻¹). The reaction mixtures were extracted with hexane (0.1 ml) byvigorous shaking.

Product Identification

The reaction products were analyzed by GC using a Hewlett Packard 6890chromatograph equipped with a Hewlett Packard 5973 mass spectrometer anda ZB-Wax plus capillary column (250 μm diameter by 30 m length, 0.25 μmfilm thickness, Phenomenex, Torrance, Calif., USA). For analysis, 1 μlof the hexane extract was injected into the GC-system. GC was performedusing various temperature profiles in dependence of the analyte.Propene: 30° C. hold 2 min; 1-butene: 35° C. hold 3.5 min, 30° C. min⁻¹to 100° C.; 1-pentene: 50° C. hold 3.5 min, 40° C. min⁻¹ to 115° C.;1-hexene, 2-hexene and 3-hexene: 70° C. hold 3.5 min, 40° C. min⁻¹ to135° C.; 1-heptene: 90° C. hold 3.5 min, 40° C. min⁻¹ 145° C.; 1-octene:110° C. hold 3.5 min, 20° C. min⁻¹ to 120° C.; +/−limonene: 70° C., 5°C. min⁻¹ to 125° C., 30° C. min⁻¹ to 230° C.; cis/trans-2-butene,isobutene and 1,3-butadiene: 35° C. hold 10 min; cyclopentene andcycloheptene: 45° C. hold 3 min, 20° C. min⁻¹ to 250° C.; isoprene: 45°C. hold 5 min, 10° C. min⁻¹ to 100° C. Helium was the carrier gas, inall cases, at a column flow rate of 1.5 ml min⁻¹. The products wereidentified relative to authentic standards by their retention timesand/or by electron impact MS at 70 eV.

Chiral Separation

The chiral separation of epoxides was performed by GC/MS using the aboveapparatus but fitted with a Beta DEX™ 120 capillary column (250 μm indiameter by 30 m length, 0.25 μm film thickness, Supelco, Bellefonte,Pa., USA). GC was performed using various temperature profiles.1-hexene: 45° C. hold 20 min; 1-heptene: 45° C. hold 37 min; 1-octene:55° C. hold 46 min; 1-octyne: 45° C. hold 2 min, 10° C./min to 155° C.The products were identified relative to authentic standards by theirretention time and by electron impact MS at 70 eV.

Product Quantification

Quantitative analyses of the reaction products were performed by GC/MSas described above, using external standard curves of the respectiveauthentic standards. All standard curves had linear regression values ofR²>0.98.

Results

TABLE 1 A “nd” means that the reaction was carried out and the productwas identified, but the product yield was not determined. Total AlcoholAmount Epoxide Amount Product Epoxide No. Substrate Product (μM) Product(μM) (μM) (%) 1

— —

117 117 100 2

20

60 80  75 3

28

12 40  31 4

6

6 12  50 5

1

9 10  88 6

88

106 194  55 7

— —

989 989 100 8

— —

496 496 100 9

— —

900 900 100 10

— —

1910 1910 100 11

— —

912 912 100 12

247

460/309 1020  76 (46/30) 13

163

561/360 1084  85 (52/33) 14

— —

nd nd nd 15

— —

nd nd nd 16

nd nd

nd nd nd 17

nd nd

nd nd nd 18

nd nd

nd nd nd 19

nd nd

nd nd nd

TABLE 1 B Substrate IUPAC name of the respective epoxide products No.(the formulae are given in column 5 of table 1 A) 1 2-methyloxirane 22-ethyloxirane 3 2-propyloxirane 4 2-butyloxirane 5 2-pentyloxirane 62-hexyloxirane 7 2,2,3-trimethyloxirane 8 2,2,3,3-tetramethyloxirane 9(2S,3S)-2,3-dimethyloxirane 10 (2R,3S)-2,3-Dimethyloxirane 112,2-dimethyloxirane 12 (4R,6R)-1-methyl-4-(prop-1-en-2-yl)-7-oxabicyclo[4.1.0]heptane/2-methyl-2-(4- methylcyclohex-3-en-1-yl)oxirane13 (4S,6R)-1-methyl-4-(prop-1-en-2-yl)-7-oxabicyclo[4.1.0]heptane/2-methyl-2-(4- methylcyclohex-3-en-1-yl)oxirane14 2-ethenyloxirane 152-ethenyl-2-methyloxirane/2-(prop-1-en-2-yl)oxirane 162-methyl-3-propyloxirane 17 2,3-diethyloxirane 186-oxabicyclo[3.1.0]hexane 19 8-oxabicyclo[5.1.0]octane

Example 2 Epoxidation of 1-Octene Using Different Fungal Peroxygenases

The peroxygenases shown in Table 2 were used in Example 2.

TABLE 2 Name Source organism Amino acid sequence Peroxygenase 1Coprinopsis cinerea SEQ ID NO: 2 Peroxygenase 2 Chaetomium virescens SEQID NO: 7 Peroxygenase 3 Humicola insolens SEQ ID NO: 8 Peroxygenase 4Chaetomium globosum SEQ ID NO: 9 Peroxygenase 5 Daldinia caldariorum SEQID NO: 14 Peroxygenase 6 Myceliophthora fergusii SEQ ID NO: 15Peroxygenase 7 Myceliophthora hinnulea SEQ ID NO: 16 Peroxygenase 8Thielavia hyrcaniae SEQ ID NO: 18 Peroxygenase 9 Pestalotiopsisvirgatula SEQ ID NO: 19

Reactions were carried out in capped 2 mL HPLC glass vials at thefollowing conditions: 10 mM phosphate buffer pH 6.5, 20% v/vacetonitrile, 1 mM 1-octene, 0.01 mg/mL peroxygenase (see Table 3), 1 mMhydrogen peroxide, in a total reaction volume of 800 μL. Reactionmixtures were stirred by magnet at room temperature (˜25° C.) for 30minutes, and the reaction was stopped by adding 5 μL catalase (TerminoxUltra 50 L, Novozymes).

Samples were extracted by mixing with 760 μL ethyl acetate containing0.01% w/v BHT (2,6-di-tert-butyl-4-methylphenol). The ethyl acetateextracts were analyzed by GC-MS on a gas chromatograph (model 7890A)equipped with an autosampler (model 7693A) and a mass selective detector(model 5975C) from Agilent (Santa Clara Calif., USA) as follows: sampleswere injected in split mode (10:1) on a Zebron DB-5HT Inferno column (15m, 250 μm, 0.25 μm) from Phenomenex (Torrance Calif., USA) and elutedwith 1.1 mL/min Helium using the following temperature program: 45° C.(for 1 min), 45-75° C. at 10° C./min, 75-275° C. at 40° C./min and 275°C. (1 minute). The mass detector was operated in scan mode and the TotalIon Count (TIC) chromatogram was used to determine the areas of thesubstrate and product peaks. Peaks were identified by comparing the massspectra with spectra from the mass spectral library available in the GCsoftware (NIST MS search version 2.0). Substrate and productconcentrations were determined by external calibration with authenticcompounds using BHT (2,6-di-tert-butyl-4-methylphenol) as internalstandard.

The enzymatic conversion of 1-octene (substrate) to 1,2-epoxyoctane(product) is shown in Table 3. Due to the volatility of 1-octene, alarge part of the substrate was lost by evaporation during the reaction.

TABLE 3 Concentration of remaining substrate and reaction product whenreaction was stopped. Concentration of Concentration of 1-octene1,2-epoxyoctane Enzyme (mM) (mM) Peroxygenase 1 0.07 0.02 Peroxygenase 20.09 0.02 Peroxygenase 3 0.07 0.19 Peroxygenase 4 0.10 0.01 Peroxygenase5 0.10 0.01 Peroxygenase 6 0.07 0.02 Peroxygenase 7 0.12 0.02Peroxygenase 8 0.09 0.04 Peroxygenase 9 0.12 0.01

Example 3 Epoxidation of Different Alkenes Using a Humicola Peroxygenase

Reactions were carried out in capped 2 mL HPLC glass vials at thefollowing conditions: 10 mM phosphate buffer pH 6.5, 20% v/vacetonitrile, 1 mM substrate (see Table 4), 0.01 mg/mL of peroxygenase 3(see Example 2), 1 mM hydrogen peroxide, in a total reaction volume of800 μL. Reaction mixtures were stirred by magnet at room temperature(˜25° C.) for 30 minutes, and the reaction was stopped by adding 5 μLcatalase (Terminox Ultra 50 L, Novozymes).

Samples were extracted by mixing with 760 μL ethyl acetate containing0.01% w/v BHT (2,6-di-tert-butyl-4-methylphenol) as internal standard.The ethyl acetate extracts were analyzed by GC-MS as described inExample 2. Due to lack of authentic standards and high volatility ofsome substrates, product yields were calculated as the Area/Aistd ofproduct in the sample compared to the Area/Aistd of a standard samplewith 1 mM substrate dissolved in ethyl acetate containing 0.01% w/v BHT,assuming the same response factor for substrate and products. Resultsare shown in Table 4.

TABLE 4 Results for epoxidation of different alkenes using Peroxygenase3. Substrate Main product Product yield 1-Octene 1.2-epoxyoctane 15%trans-2-Octene 2,3-epoxyoctane 63% trans-3-Octene 3,4-epoxyoctane 69%trans-4-Octene 4,5-epoxyoctane 76% 1,9-Decadiene 1,2-epoxy-9-decene 13%

Example 4 Epoxidation of Trans-2-Octene Using a Humicola Peroxygenase

Reactions were carried out in capped 2 mL HPLC glass vials under thefollowing conditions: 10 mM phosphate buffer pH 6.5, 20% v/vacetonitrile, 20 g/L trans-2-octene, 0.1 or 0.5 mg/mL of peroxygenase 3(see Example 2), 160 μL of 1 M hydrogen peroxide dosed during thereaction (2 hours) with a multi-channel syringe pump (model 220-CE,World precision instruments, Aston, Stevenage, UK) using 1 mL gas tightglass syringes (SGE Analytical Science, Ringwood, Australia); in a finalreaction volume of 800 μL. Reaction mixtures were stirred by magnet atroom temperature (˜25° C.) for 2 hours, and the reaction was stopped byadding 5 μL catalase (Terminox Ultra 50 L, Novozymes).

Samples were extracted by mixing with 780 μL ethyl acetate containing0.1% w/v BHT (2,6-di-tert-butyl-4-methylphenol) as internal standard.The ethyl acetate extracts were diluted 100 times with ethyl acetatewithout internal standard and analyzed by GC-MS as reported in Example2. Formation of epoxide was measured as the area percentage of epoxidecompared to the area sum of substrate and products, assuming the sameresponse factor for substrate and product.

TABLE 5 Results from epoxidation of trans-3-octene using Peroxygenase 3.Peroxygenase concentration Yield of 2,3-epoxyoctane   0 mg/mL 0% 0.1mg/mL 3% 0.5 mg/mL 40% 

1-15. (canceled)
 16. A method for producing an epoxide, comprisingcontacting a non-cyclic aliphatic alkene or a terpene with hydrogenperoxide and a peroxygenase (EC 1.11.2.1), wherein the peroxygenasecomprises an amino acid sequence having at least 90% sequence identityto SEQ ID NO:
 16. 17. The method of claim 16, wherein the peroxygenasecomprises an amino acid sequence having at least 95% sequence identityto SEQ ID NO:
 16. 18. The method of claim 16, wherein the peroxygenasecomprises an amino acid sequence having at least 97% sequence identityto SEQ ID NO:
 16. 19. The method of claim 16, wherein the peroxygenasecomprises an amino acid sequence having at least 98% sequence identityto SEQ ID NO:
 16. 20. The method of claim 16, wherein the peroxygenasecomprises an amino acid sequence having at least 99% sequence identityto SEQ ID NO:
 16. 21. The method of claim 16, wherein the peroxygenasecomprises an amino acid sequence having the amino acid sequence of SEQID NO:
 16. 22. The method of claim 16, wherein the amino acid sequencecomprises the motif E-H-D-[G,A]-S-[L,I]-S-R (SEQ ID NO: 21).
 23. Themethod of claim 16, wherein the aliphatic alkene has one or moresubstituents selected from the group consisting of halogen, hydroxyl,carboxyl, amino, nitro, cyano, thiol, sulphonyl, formyl, acetyl,methoxy, ethoxy, carbamoyl and sulfamoyl.
 24. The method of claim 23,wherein the substituent(s) are selected from the group consisting ofchloro, hydroxyl, carboxyl and sulphonyl.
 25. The method of claim 16,wherein the aliphatic alkene consists of at least three carbons, and hasa carbon-carbon double bond at one end.
 26. The method of claim 16,wherein the aliphatic alkene is propene, butene, pentene, hexene,heptene, octene, nonene, decene, undecene, dodecene, tridecene,tetradecene, pentadecene, or hexadecene, or an isomer thereof.
 27. Themethod of claim 16, wherein the aliphatic alkene is propene, 1-butene,1-pentene, 1-hexene, 2-hexene, 3-hexene, 1-heptene, 1-octene,2-methyl-2-butene, 2,3-dimethyl-2-butene, cis/trans-2-butene, isobutene,1,3-butadiene, and isoprene; or an isomer thereof.
 28. The method ofclaim 16, wherein the aliphatic alkene is unsubstituted.
 29. The methodof claim 16, wherein the aliphatic alkene is linear.
 30. The method ofclaim 16, wherein the terpene is isoprene or a monoterpene.
 31. Themethod of claim 16, wherein the terpene is a cyclic terpene.
 32. Themethod of claim 31, wherein the cyclic terpene is a monocyclicmonoterpene.
 33. The method of claim 32, wherein the monocyclicmonoterpene is limonene.